Chronic myeloid leukemia (CML) is characterized by hyperproliferation of myeloid progenitors that retain the capacity for differentiation. The natural history of CML involves progression to a more aggressive disease blast crisis phase, where terminal differentiation is impaired, and the disease exhibits features of acute leukemia. Much like many of the other known myeloproliferative disorders, CML is associated with an activated tyrosine kinase. In the case of CML, the fusion protein, BCR-ABL, which is produced following a reciprocal chromosomal translocation event between chromosomes 9 and 22 is causative of the disease. Current treatment for CML involves the use of tyrosine kinase inhibitors (TKI), which have been shown to result in a deep clinical remission in the majority of cases. However, the emergence of resistance-conferring mutations preventing TKI binding in the ATP pocket is frequently responsible for disease relapse. Second and third generation TKIs have been developed which collectively retain activity against all resistance-conferring mutations. With improved control of this on-target resistance mechanism, clinical resistance in the future will increasingly be a consequence of off-target resistance mechanisms, which are presently poorly understood. The objective of this work is to identify and validate candidate molecular mediators of off-target resistance in CML. In preliminary data involving analysis of primary samples obtained from CML patients with clinical features of BCR-ABL-independent disease and persistent MAPK and JAK/STAT signaling despite BCR-ABL inhibition, we have identified the presence of activating NRAS mutations in conjunction with EVI-1 overexpression. We therefore hypothesize that activated NRAS and EVI-1 overexpression can cooperatively confer off-target resistance to BCR-ABL TKI treatment. In Aim 1 we will use BCR-ABL-expressing cell line and murine models to assess the ability of these lesions alone and in combination to confer resistance to BCR-ABL TKIs. In Aim 2 we will assess the impact of the sequence of introduction of these mutations on differentiation and progression to a blast crisis like phenotype. Finally, in Aim 3 we will assess the impact of these lesions upon the status of critical signaling pathways. It is anticipated that this work will provide valuable insights into the nature of oncogene addiction, the phenomenon of exquisite reliance upon the activity of a particular oncogenic lesion. Additionally, it is hoped that adjunctive therapeutic targets will be identified that may improve treatment outcomes for patients with CML and other malignancies associated with pathologic activation of tyrosine kinases. PUBLIC HEALTH RELEVANCE: Chronic Myeloid Leukemia, or CML, is a potentially fatal cancer of the white blood cells. Although effective targeted treatments have been developed, the occurrence of drug-resistant disease remains a significant problem for a proportion of patients. Our research aims to identify and validate molecular mechanisms responsible for this resistance to treatment, with the eventual goal of identifying new therapeutic targets. We strongly believe that what we learn about molecular mechanisms of resistance to targeted therapies in CML will be applicable to the burgeoning number of cancers treated with targeted therapies, and will provide valuable insights into cancer biology.